Sickle cell disease (SCD) is caused by a mutant hemoglobin (Hb) molecule that polymerizes in hypoxia, leading to vaso-occlusion and hemolysis. Pulmonary Hypertension (PH), characterized by elevated pulmonary artery pressure and endothelial dysfunction, is a leading cause of death in adult SCD patients and is strongly associated with hemolysis and platelet activation. Notably, we and others have shown that hemolysis directly activates platelets, and activated platelets are known to secrete vasoactive substances, such as the glycoprotein thrombospondin-1 (TSP1), which promotes endothelial dysfunction and vascular injury. While prior studies link hemolysis and platelet activation to vascular injury, the mechanism by which hemolysis induces platelet activation, and the mediators linking platelet activation to endothelial dysfunction and PH are unknown. We recently discovered a novel mechanism of hemolysis-induced platelet activation in which Hb (released via hemolysis) inhibits platelet mitochondrial complex V, leading to the production of mitochondrial reactive oxygen species (mtROS), which activate platelets. Preliminary data now suggest a novel receptor mediated pathway in which Hb activates platelet toll like receptor 4 (TLR4) to inhibit complex V. Further, we show ADP also inhibits complex V and generates mtROS to activate platelets and release platelet TSP1. Importantly, circulating TSP1 is elevated in SCD patients. Based on these data, we hypothesize that hemolysis-induced platelet mtROS cause TSP1 release from platelets, which promotes downstream endothelial dysfunction and PH in SCD. We will test this hypothesis with three specific aims that integrate in vitro experiments in healthy and SCD human platelets, ex vivo microfluidic vessel studies, and in vivo murine models. Aim 1 will elucidate the signaling pathway by which Hb and ADP activate TLR4 and purinergic signaling to post-translationally modify platelet mitochondrial complex V, leading to mtROS-dependent platelet activation. Aim 2 will utilize bone marrow chimeric mice to determine whether TSP1 released from the platelet promotes vascular injury through interaction with its endothelial cognate receptor CD47. Aim 3 will determine whether pharmacological scavenging of mtROS or blocking of TSP1 or CD47 function attenuates PH pathogenesis in a SCD murine model. Transgenic SCD mice will be chronically treated with MitoQ (a mitochondrial targeted antioxidant), TSP1 blocking antibody, or anti-CD47 blocking antibody and markers of endothelial dysfunction and PH assessed. Ex vivo microfluidics will be used to dissect the effects of MitoQ on platelets versus the endothelium. This project will elucidate a novel receptor-mediated signaling axis by which hemolysis induces vascular injury and PH. Further, pre-clinical testing will establish mtROS and the TSP1- CD47 axis as viable therapeutic targets in SCD-PH and lay the groundwork for future clinical trials to repurpose MitoQ and anti-CD47 antibody (both currently in Phase II clinical trials for indications) for SCD-PH.